The overarching goal of this U19 Program is to use forward genetic screens in mice to advance our understanding of immune responses to pathogens. This U19 Program is driven by our recent development of a genetic screening platform that uses pooled, in vivo CRISPR-mediated loss-of- function genetic screens to identify genes that positively or negatively regulate the fate and function of immune cells. We will couple this novel approach with single-cell gene profiling and systems-level computational modeling of innate and adaptive cells during acute infection to nominate candidates to perturb functionally in genetic screens. We will then test thousands of potential regulators in pooled CRISPR-Cas9 based forward genetic screens in mice, and validate novel candidate regulators in mouse infection models and human cells. Our proposed U19 Program consists of 2 highly interactive Projects, supported by 4 Cores. Project 1 (Haining/Kuchroo/Sharpe) will conduct forward genetic screens to identify genes that regulate the fate and function of CD8+ and CD4+ T cells responding to acute infection. Project 2 (Hacohen/Kagan) will conduct screens to identify genes that control DC activation in response to pathogens, pathogen components and T cells. Administrative Core A (Sharpe/Haining) will provide administrative and scientific coordination, and implement our Pilot Project program. Data Management and Bioinformatics Core B (Regev) will develop, apply and disseminate cutting-edge methods and tools for single cell RNA-seq analysis of immune cell responses and for selecting and ranking candidate genes for genetic manipulation in CRISPR screens. Core B will also establish and maintain a public portal and software pipelines for sharing data, analyses and methods. CRISPR Library Core C (Doench) will design and generate custom single guide RNA (sgRNA) libraries needed to conduct forward genetic screens and to validate candidate regulators. Core C will also analyze genomic DNA from cells obtained pooled screens, performing sequencing and sequence deconvolution to identify sgRNAs that caused the phenotype of interest. Mouse Perturbation Core D (Sharpe/Haining) will provide a uniform platform to execute CRISPR-Cas9 screens and validation experiments in mouse infection models. The use of standardized experimental methods and computational tools by the cores will make it possible to compare and integrate results in different settings and disease models. We expect that our in vivo forward genetic screens and systems level single-cell genomic analyses will identify the central molecules, pathways and mechanism that guide innate and adaptive immune responses to infection. These findings will lay the foundation for new vaccination strategies for infectious diseases and therapies for allergy and autoimmunity.